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Antibiotic-resistant Staphylococcus aureus: Investigation of a poultry reservoir

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  • Staph aureus is a common bacterium that can cause superficial as well as deep and systemic infections. MRSA infection may begin as a reddish rash with lesion(s) that looks like a pimple or small boil. Often it progresses to an open, inflamed area of skin (as pictured in the lower right) that may weep pus or drain other similar fluid. It can manifest into more a severe infection with symptoms of: endocarditis, necrotizing fasciitis, osteomyelitis, and sepsis. Some cases become life threatening. While the earliest history of infections with methicillin-resistant S. aureus (MRSA) typically occurred in a hospital setting in the 1970s, more current information reveals additional risk groups outside of hospitals within the broader community [a.k.a. community-acquired MRSA (1990s)]. MRSA may be found on human skin and in the nasopharynx, where they can be carried asymptomatically. MRSA is a major public health concern because infections are more difficult to treat and lead to higher mortality rates than infections with methicillin-susceptible S. aureus (MSSA). CA-MRSA also has been reported to cause infections in groups which typically are not at risk of MRSA infections, including athletes, young children, and those in the military and in correctional institutions. Community-acquired infections are becoming more common in individuals relative to HA-MRSA. It is estimated that roughly 1% of the population (~2.3 million Americans) are colonized with some type of MRSA, and that HA-MRSA was responsible for over 94,000 infections and 18,000 deaths in the U.S. in 2005. Further, a newly found MRSA type within the community is livestock-associated MRSA, making livestock producers a newly identified risk group. A 2009 study (Graham – US) indicated antibiotic-resistant S. aureus was found on flies collected near confined poultry operations, indicating another avenue of spread into the community. Further, there have been several anecdotal cases and peer-reviewed articles suggesting that farmers and hatchery workers have contracted antibiotic-resistant S. aureus in the workplace setting.
  • Antibiotic-resistant S. aureus can cause infections in a variety of animals. The bacterium has been isolated from horses, cattle, dogs and cats, and most recently, in swine. Swine have been the most heavily studied animals. It is not currently known whether humans are transient carriers or reservoirs transferring antibiotic-resistant S. aureus to animals, or whether strains of antibiotic-resistant S. aureus have entered the animal populations and may be endemic there as well (or whether both routes of transmission may have occurred, or still be occurring). Studies in swine herds have shown that isolates obtained from swine and their human caretakers are frequently indistinguishable, suggesting transmission can occur between human and animal species. The occurrence of secondary LA-MRSA infections, as exemplified by the finding of LA-MRSA infected individuals who have no known exposure to live animals, suggests transmission via human carriers or by exposure to contaminated environments. Investigations in the Netherlands have shown that a strain of MRSA that was first found in swine and cattle (referred to by its molecular type, ST398) now accounts for 20% of human MRSA cases in that country. Thishighlightsthe importance of considering livestock and other animals in antibiotic-resistant S. aureus epidemiology. Currently the prevalence of antibiotic-resistant S. aureus in poultry or their caretakers is unknown in the U.S. I am proposing to carry out the first examination of the prevalence of S. aureus in live poultry and poultry workers in the U.S. LA-MRSA has been documented in an increasing number of countries across Europe, Asia, and North America. However, most of these studies have focused on pigs and cattle, and livestock veterinarians, with very few studies carried out in poultry, and none in live turkeys. In a rural state such as Iowa – which is the nation’s leader in egg production and produces 8.2 million turkeys per year – antibiotic-resistant S. aureus on poultry farms could complicate efforts to reduce transmission statewide.
  • In most environments, transmission of antibiotic-resistant S. aureus takes place either through direct person-to-person contact (for example, during skin contact) or via contaminated fomites such as doorknobs. However, in an environment with several thousand antibiotic-resistant S. aureus-colonized animals concentrated into a relatively limited space, a greater proportion of the bacteria have the potential to dry on environmental surfaces and become aerosolized due to animal movement and the high-powered ventilation systems. Therefore, airborne transmission is likely to play a large role in colonization of both animals and humans inside the barns. Previous studies have examined the presence of antibiotic resistant bacteria within and outside of animal barns. One study examined airborne bacteria within and downwind of two confined animal feeding operations (CAFOs), but did not test for the presence of antibiotic-resistant S. aureus. Other environmental samples were not tested, and bacterial loads were only measured up to 25 m outside of the barns. A 2006 study repeated this, sampling up to a distance of 150 m downwind, but still did not examine MRSA. Additionally, both studies used two-stage Andersen samplers rather than six-stage samplers (six stage samplers provide a better definition of the source of the particles that contain MRSA). Although our group has identified LA-MSSA in approximately 25% of retail turkey meat samples in Iowa, and LA-MRSA was found in over 35% of retail turkey meat samples and 16% of chicken samples in the Netherlands, and in broiler chickens in Belgium, a study of LA-MRSA in live poultry has not been conducted in the United States. Several studies have suggested the occurrence of secondary spread of LA-MRSA as exemplified by the finding LA-MRSA infected individuals who have no known exposure to live animals, suggesting transmission via human carriers or by exposure to contaminated environments or food products. Greater knowledge of the ecology and epidemiology would is invaluable in assessing infectious disease risks and providing recommendations for preventative measures.
  • Our long-term goalis to better understand the ecology and epidemiology of antibiotic-resistant S. aureus, including MRSA, in the community environment, specifically in the agricultural environment. Our objective in this application is to characterize the ecology and epidemiology of S. aureus associated with poultry farming. Our central hypothesisis that individuals working in close proximity to poultry are at risk of occupational exposure to antibiotic-resistant S. aureus. Further, we hypothesize that farmers in contact with poultry will be colonized with livestock-associated S. aureus strains previously found in live chickens and retail meat samples.The proposed research is innovative because it is designed to examine the epidemiology of S. aureus in an agricultural/rural setting, rather than in the health care setting where most research has been carried out to date. Additionally, we are proposing the first study of S. aureus in live turkeys and turkey workers, and the first study of this in chickens in the United States.
  • We will test our central hypothesis and accomplish the objective of this application by pursuing the following specific aims: Establish the prevalence and molecular characteristics of antibiotic-resistant S. aureus on poultry farms. Our working hypothesis is that poultry will be colonized with antibiotic-resistant S. aureus; and that individuals working with live animals will be colonized with identical strains of this bacterium. We will collect nasal and pharyngeal swabs of workers and poultry on selected poultry farms. We will culture the samples on selective media, and carry out molecular typing of S. aureus isolated from these samples. Establish the prevalence and molecular characteristics of antibiotic-resistant S. aureus in poultry processing facilities. Our working hypothesis is that poultry will be colonized with antibiotic-resistant S. aureus; and that areas where animals are slaughtered and processed will be contaminated with identical strains of this bacterium. We will collect surface swabs of processing facilities, poultry, feed, litter, and eggs at selected poultry processing facilities. We will also collect air samples throughout the processing facility. We will culture the samples on selective media, and carry out molecular typing of S. aureus isolated from these samples. Determine risk factors for presence of antibiotic-resistant S. aureus on farms or processing facilities.Our working hypothesisis that the presence of antibiotic-resistant S. aureus on farms will be associated with use of antibiotics during animal husbandry. After aim 1 is completed, questionnaire data will be analyzed using statistical software.
  • Livestock-associated MRSA (LA-MRSA) has been documented in an increasing number of countries across Europe, Asia, and North America, although most studies have examined a relatively small number of farm workers in cross-sectional studies and have frequently been conducted on the farm site. However, such epidemiologic studies in poultry have not yet been conducted in the United States. A cross-sectional study examining carriage and transmission of antibiotic-resistant S. aureus of poultry, poultry workers, and poultry farms is of vital importance, as the epidemiology of MRSA appears to be rapidly changing to a more apparent public health issue. For example, community-associated MRSA strain USA300, first reported in 1998 in North Dakota, was recently found in 29% of invasive MRSA isolates collected from US metropolitan areas, demonstrating how quickly new strains can become established in the population. Therefore, there is a critical need to better understand the epidemiology of novel S. aureus isolates associated with farming exposures. Such knowledge would be invaluable in assessing infectious disease risks and providing recommendations for preventative measures.While several studies have been conducted on antibiotic-resistant S. aureus in swine and cattle, there have been few studies pertaining to poultry. Two studies have examined antibiotic-resistant S. aureus in chickens from Belgium. However, the studies did not include workers. Furthermore, no studies have investigated antibiotic-resistant S. aureus in turkeys or turkey farmers.
  • In 2008 and 2009, we sampled a total of 18 swine farming systems in Iowa and Illinois. Nine of these were confined animal feeding operations (CAFOs), which typically have large numbers of animals (hundreds to thousands of animals per barn) and administer antibiotics throughout the lifespan of the animal. An additional 9 organic/antibiotic-free swine farms were also sampled, but MRSA was not detected on these farms. Five of 18 (27.7%) of farms were found to be positive for MRSA; 55.5% of confinement operations tested were positive (5/9). Overall prevalence in swine was 10% (43/428 animals). Overall, the prevalence of MRSA in humans was 32.6%. However, in humans that worked in confinement operations, the prevalence was higher at 45.3% (29/64).
  • We will be collaborating with the Iowa State University to examine multiple poultry production systems, including broiler, layer chickens, and turkeys. All farms examined will be in the state of Iowa. Given numbers from previous research in live chickens, we estimate a 40% (± 5.0%) prevalence of S. aureus in live birds. Using a 95% confidence interval and 80% power, we calculated the sample size of live animals to be 600. To reach sufficient sample size, we will be conducting convenience sampling of 20 animals from 30 different farms (10 turkey farms, 10 layer farms and 10 broiler farms). At each farm, all consenting caretakers will also be sampled (estimating 1-2 employees per farm, totaling 30-60 humans). We are looking for zoonotic transmission and therefore identical strains between the humans and live poultry.
  • We have gained IRB-01 approval for this study. Humans are typically colonized with S. aureus in the anterior nares, and nasal swabs are commonly used for detection of MRSA carriers. However, nasal swabs alone miss approximately 13% percent of MRSA carriers; therefore, we will employ both nasal and throat swabs for this study in order to maximize sensitivity of detection. A study subject will be considered to have occupational exposure to livestock/poultry when his or her employment duties involve entering buildings where live animals are housed. Such building entry must occur on average one or more times a week over the previous year. Slaughterhouse workers may have such exposures if their worksite is located prior to the kill room. Further, a comparison group has been established from an ongoing pilot study of the prevalence of S. aureus in rural Iowa (SIRI). The comparison group, recruited in rural Iowa, has no exposure to livestock or poultry. Human subjects will be sampled once over the course of the research.ICAUC approval has been obtained. Poultry will be swabbed in a similar manner. Samples will be collected from the choanal (palatine) cleft and from the cloaca of the selected animal. Sterile swabs will be inserted approximately 1 cm into the choanal cleft and rotated against the anterior mucosa. The swab will then be returned to the tube for transportation. Sterile swabs will also be inserted approximately 2 cm into the cloaca of the selected animal and returned to the tube for transportation. Samples will be collected by the production system’s veterinarians, caretakers, and trained laboratory staff. Animals will be sampled once over the course of the research.The six-stage Andersen viable cascade impactor was developed to allow simultaneous sizing and counting of viable microorganisms in air samples. It has the advantage of collection directly onto culture media for incubation and analysis with no dilution or plating of organisms. Air will be sampled internally in barns and processing facilities described. Samplers will be placed at breathing zone level approximately in the center of the work area when possible.
  • Given numbers from previous research in chicken and turkey meat, we estimate a 25% prevalence of S. aureus in poultry processing facilities. Samples will be collected from participating poultry processing facilities in Iowa. We will collect surface swabs of processing facilities, poultry, feed, litter, and eggs at selected poultry processing facilitiesA questionnaire will be administered, focused upon capturing animal exposure data in the previous year, as well as capturing demographic and some behavioral data. The enrollment questionnaire covers areas including general demographic data such as age and race, as well as study-specific questions such as the number of animals the subjects are in contact with, what type of work they perform with live animals, contact hours/years with animals, use of gloves or other personal protective equipment while working, smoking (whether at during work hours or not), presence of any cuts or abrasions on the skin in the prior nine months, and additional risk factors that we hypothesize may predispose to infection with antibiotic-resistant S. aureus (including use of antibiotics in the past three months; participation in team sports; or exposure to correctional facilities. In addition, the questionnaire will ask whether participants or their family members work in healthcare facilities including hospitals and long-term care facilities, or whether they have been hospitalized in the previous 12 months, and whether they have had any skin or soft tissue infections or antibiotic-resistant S. aureus infections in the previous 12 months). A questionnaire will be administered to the present administrator or manager and will focus upon capturing farm-specific data. The farm questionnaire covers study-specific questions including the numbers of animals, the source of poultry (eggs or chicks), feed source, litter type and how often it is changed, if the eggs are washed or unwashed, type of poultry (breed and origin), how long the farm has been populated, and what, if any, antibiotics are used. In addition, the questionnaire will ask general questions including how many employees work on the farm, what the turnover rate is like, whether farm workers are offered ammonia-filtered masks, if the masks are fit-tested, and the approximate compliance rate among workers.
  • Samples will be collected using sterile swabs and inserted into liquid Stuart’s medium for transportation to the Emerging Pathogens Laboratory at the Center for Emerging Infectious Diseases. Samples collected by investigators will be stored on ice until transport and processing (within 48 hours). Swab samples will be inoculated into 5mL enrichment broth. Cultures will be incubated for 24h at 35°C, then 5 µL of broth will be inoculated onto selective MRSA agar plates and onto Columbia CNA agar plates (CNA). Isolates will be confirmed as S. aureus by examining their appearance on Gram stain, and by doing the catalase test, the coagulase test and the S. aureus latex agglutination assay. Methicillin resistance will be confirmed by testing for the presence of penicillin binding protein (PBP2). Antibiotic-resistant S. aureus isolates will be stored at -80°C. Positive and negative controls will be used for all tests.
  • All isolates will be analyzed by spa typing (a PCR/sequencing method which distinguishes between closely related isolates); pvl PCR (which detects the presence of the Panton-Valentine Leukocydin toxin, a potential virulence factor), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and antibiotic resistance testing as described below. Isolates representing distinct spa patterns on each farm will be subjected to further typing. If all isolates on an individual farm show identical spa patterns, 5 isolates will be randomly selected for further analysis. This will allow us to minimize duplicative testing of closely related isolates that are unlikely to significantly increase our knowledge of the epidemiology of this organism. However, all isolates will be retained and frozen down for potential future studies. Isolates chosen for further analysis will be subjected to MLST typing. A subset of unique S. aureus isolates will be tested in the hospital laboratory for antimicrobial susceptibility by the broth dilution method described by the Clinical and Laboratory Standards Institute. Isolates will be tested for susceptibility to tetracycline, erythromycin, clindamycin, trimethoprim-sulfamethoxazole, gentamicin, levofloxacin, moxifloxacin, linezolid, daptomycin, vancomycin, and rifampin.
  • All data from prevalence and molecular tests will be independently double-entered into databases and checked to ensure accuracy. Questionnaire and laboratory data will be linked by a unique specimen number. Multivariate modeling of risk factors will be performed using logistic regression. A trend in prevalence of antibiotic-resistant S. aureus in poultry by type will be tested with the Cochran-Armitage trend test. A significance level of 0.05 will be used in the analyses. Analyses will be performed using SAS software version 9.1 (SAS Institute Inc., Cary, NC).
  • The proposed study has several strengths, including being the first study of S. aureus in live turkeys and turkey workers, and the first study of this in chickens and chicken workers in the United States. We have a well-qualified, interdisciplinary research team consisting of investigators with expertise in microbiology, environmental health, occupational and rural medicine, veterinary medicine, and epidemiology. Further, the state of Iowa is a national leader in eggs and turkey production.Limitations also exist in the study. Because farms are convenience, single-time samples, they may not be representative of all farms. Additionally, the proposed research is a pilot study, indicating a potential need for further research. Also, the limited geography of the study area many not be representative of the United States in general.
  • Greater knowledge of the ecology and epidemiology of these organisms is invaluable in assessing infectious disease risks and providing recommendations for preventative measures. At the completion of this research, it is our expectation that we will have established the prevalence and dominant molecular types of S. aureus in poultry and poultry workers, all of which provide novel routes of transmission for this bacterium. Such results will have an important positiveimpact on the field, because they will provide insight into the maintenance and spread of S. aureus in the rural environment, as well as potential mechanisms that could be implemented to prevent such emergence and spread.
  • Transcript

    • 1. DISSERTATION PROPOSAL: Antibiotic-resistant Staphylococcus aureus: Investigation of a poultry reservoir Department of Occupational & Environmental HealthSept.10, 2010 Abby L. H. Maples, MPH
    • 2. Introduction Background  Significance Goals Specific Aims Limitations of Current Literature & Research Research Design & Methods Strengths & Limitations
    • 3. Background Staphylococcus aureus  Signs & symptoms  Methicillin-resistant S. aureus (MRSA)  Hospital  94,000 infections (Klevens, 2007)  18,000 deaths (Klevens, 2007)  Community  1% of US population colonized (Graham, 2006; Kuehnert, 2006; Shorr, 2007)  Livestock http://www.medicinenet.com/mrsa_infection/page2.
    • 4. Antibiotic-resistant S. aureus &Animals S. aureus can cause infection in animals (Baptiste, 2005; De Neeling, 2007; Juhasz- Kaszanyitzky, 2007; Khanna, 2008; Smith,2009; Van Loo, 2007; Weese, 2005; Weese,2006) Role of humans  Transient carriers or reservoirs? Role of animals  Endemic? Swine herds studied Livestock-associated MRSA increasing
    • 5. Antibiotic-resistant S. aureus inEnvironmental Samples Airborne transmission  Colonization role? Previous airborne transmission studies (Gibbs, 2004; Gibbs, 2006) Surface contamination Meat contamination (De Boer, 2009; Hanson, in press) http://www.takepart.com/news/tag/anti-factory- farming
    • 6. Goals Long-term goal  Understand ecology & epidemiology of antibiotic- resistant (AbR)S. aureus Objective  Characterize ecology & epidemiology of AbR S. aureus Central hypothesis  Workers in close proximity to poultry are at risk of occupational exposure to AbR S. aureus  Farmers in contact with poultry will be colonized with MRSA
    • 7. Specific Aims1. Establish the prevalence & molecular characteristics of AbR S. aureus on poultry farms2. Establish the prevalence & molecular characteristics of AbR S. aureus in poultry processing facilities3. Determine risk factors for presence of AbR S. aureus on farms or processing facilities
    • 8. Limitations of Current Literature S. aureus is rapidly changing Previous studies in chickens did not include workers Epidemiologic studies in poultry have not been conducted in U.S. http://www.sciencedaily.com/releases/2009/03/090316120846.htm
    • 9. Preliminary Studies Prevalence of MRSA in Iowa & Illinois swine 18 swine farms in Iowa & Illinois (Smith, 2009) 9 each – confined animal feeding operations (CAFOs) & antibiotic-free systems 5/18 (27.7%) farms positive for MRSA  All CAFOs (55.5%, 5/9) Prevalence in swine 43/428 (10.0%) Prevalence in humans 29/89 (32.6%)  Humans working at CAFOs (45.3%, 29/64)
    • 10. Research Design & Methods Farm Description  Broilers, layers, and turkeys  Estimate 40% (± 5.0%) prevalence in poultry  95% confidence interval, 80% power = 600 animals  20 animals from 30 farms (10 each)  Convenience Sample  Estimate 1-2 employees per farm  Goal to enroll 60 humans http://newshopper.sulekha.com/turkey-farm_photo_1063248.
    • 11. Research Design & MethodsContinued Sample Collection  IRB& IACUC Approved  Humans  Nasal & throat Swabs  Comparison, non-exposed group  Poultry  Choanal (palatine) cleft & cloaca swabs Air Sample Collection  6-stage Andersen Viable Cascade Impactor
    • 12. Research Design & MethodsContinued Processing Facility Sample Collection  Estimate 25% prevalence in processing facilities  Surface & worker swabs of facility Occupational Exposure Questionnaire  Animal exposure in last year  Demographic & behavioral questions Farm Owner/Manager Questionnaire  Farm-specific questions
    • 13. Research Design & MethodsContinued Bacterial Growth  6.5% Staph enrichment broth, CNA & CHROMagar Microbiology  Gram stain, catalase, coagulase, S. aureus latex agglutination assay  MRSA confirmed by penicillin binding protein presence
    • 14. Diagnostic Schematic
    • 15. Research Design & MethodsContinued Molecular Typing  Spa typing  PVL PCR  Multilocus sequence typing (MLST)  Pulsed-Field Gel Electrophoresis (PFGE) Antibiotic Resistance Testing  Brothdilution (CLSI standards)  Tested for susceptibility
    • 16. Research Design & MethodsContinued Data and Statistical Analysis  Double-entry into databases  Excel, Access, and Teleform Verifier  Unique specimen numbers  Multivariate modeling of risk factors (SAS)  Logistic regression  Cochran-Armitage trend test
    • 17. Strengths & LimitationsStrengths Limitations First study of S. aureus  Convenience, single- in live poultry & workers time samples (not in US representative) Well qualified &  Pilot study experienced team  Limited geography Collaboration with ISU  Limited farmer Iowa is a national leader enrollment in eggs and turkey production
    • 18. Impact & Benefit to Field Assess infectious disease risks Provide recommendations for preventative measures Establish prevalence and dominant molecular types of S. aureus in poultry Create potential implementations to prevent spread of S. aureus in rural Iowa
    • 19. Three Publishable Paper Topics Literature review of poultry diseases Antibiotic resistant Staphylococcus aureus: Investigation into a poultry reservoir Staphylococcus aureus in environmental samples and humans: Two poultry production facilities in Iowa(?) Airborne Staphylococcus aureus in (10) poultry barns (?)**Open for discussion
    • 20. Project Timeline  Oct 2010 – Sept 2011: Farm Sampling  Dec 2010 – June 2012: Molecular analysis & antibiotic resistance testing  June – Sept 2012: Finish analysis & draft manuscript April- Jan-Oct-Dec Jan-March July-Sept Oct-Dec April-June July-Sept June March2010 2011 2011 2011 2012 2012 2011 2012Farm Sampling Molecular analysis & antibiotic resistance testing Finish analysis & draft manuscript
    • 21. Questions, Comments, or Concerns?http://ehsmanager.blogspot.com/2009/05/bacteria-create-aquatic-superbugs-

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